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Abstract

Background

The novel pandemic A (H1N1) pdm09 virus was first identified in Mexico in April 2009
and since then it spread worldwide over a short period of time. Although the virus
infection is generally associated with mild disease and a relatively low mortality,
it is projected that mutations in specific regions of the viral genome, especially
within the receptor binding domain of the haemagglutinin (HA) protein could result
in more virulent virus stains, leading to a more severe pathogenicity.

Methods

To monitor the genetic polymorphisms at position 222 of Haemagglutinin of influenza
A(H1N1)pdm09 viruses from both outpatients with mild influenza and individuals with
severe disease requiring hospitalization, during 2009–2010 and 2010–2011 seasons,
a sequence-based genotypic assessment of viral populations to understand the prevalence
of D222G mutation.

Results

The D222G was identified in clinical specimens from 3 out of 42 cases analyzed in
Tunisia with severe outcome (7%). Interestingly, in one fatal case out of four viruses
taken from fatal cases studied (25%). Also this mutation was found in one mild case
out of 8 mild cases studied (0.1%). D222E substitution was found in virus taken from
one patient with severe clinical syndrome (2%) out of 42 severe cases analyzed and
E374K substitution was found in two severe cases (4%) out of 42 severe cases studied.

Conclusions

A specific mutation in the viral haemagglutinin (D222G) was found in fatal, severe
and mild case. Further virological, clinical and epidemiological investigations are
needed to ascertain the role of this and other mutations that may alter the virulence
and transmissibility of the pandemic influenza A (H1N1)pdm09.

Keywords:

Introduction

In April 2009, a novel swine-derived influenza A(H1N1)pdm09 emerged and spread rapidly
around the world [1,2], causing the World Health Organization to declare a pandemic in June. Since the first
appearance of influenza A(H1N1)pdm09, one particular amino acid substitution {aspartic
acid to glycine at position 222 (D222G)} (225 in H3 numbering) within the hemagglutinin
(HA) molecule has appeared sporadically in 20 countries, including Norway, Mexico,
Ukraine and the USA [3-5]. The D222G substitution is known to cause a shift from α2,6-SA receptor specificity
to mixed α2,3/α2,6-sialic acid receptor specificity [6,7]. It is noteworthy that is highly conserved among avian viruses [8]. Previously, α2,3-specific avian viruses have been isolated from patients during
the initial phases of the pandemics of 1957 and 1968, and avian HA in humans has been
shown to be selected for increased affinity for the α2,6 receptor [8]. Also, the substitution was present in the Spanish Flu outbreak of 1918 [9]; however, the existence and transmissibility of influenza A(H1N1)pdm09 α2,3-SA specific
viruses remain unclear. To identify whether α2,3-SA specific viruses, which replicate
well in swine, were spread during the early phase of the pandemic and whether α2,3-SA
specific viruses are easily transmitted, the nucleotide sequences of the HA receptor
binding site of influenza A(H1N1)pdm09 in clinical specimens were determined in this
study.

In an attempt to understand the relevance of HA D222G substitution among influenza
A(H1N1)pdm09 causing infections in Tunisia, HA gene sequences from respiratory specimens
of severe and non-severe cases were examined. In addition to the D222G substitution,
we focused on another substitution {glutamic acid 374 to lysine acid (E374K)}, mutation
located at the stalk of HA2 in the cavity where the fusion domain of mature HA molecules
might have an impact on the antigenicity or neutralization activity of influenza A(H1N1)pdm09
[10-12].

Material and methods

Nasopharyngeal or throat swab specimens from influenza patients are received directly
from sentinel primary care physicians participating in virological surveillance schemes
in the community. Samples are also received from community, hospitalised and fatal
cases are forwarded to the Tunisia National Influenza Centre for diagnostic and further
characterisation. A total of 7350 specimens from influenza patients were collected
in Tunisia during 2009–2010 season and 894 specimens during 2010–2011 season.

The samples used in this study were taken from 50 patients including 42 respiratory
specimens from severe (patients hospitalized with severe pneumonia and severe acute
respiratory syndrome) and fatal cases, as well as from 8 cases with mild clinical
outcomes. Mild cases presented with at least one of the following influenza-like illness
symptoms: fever of at least 38°C, cough, rhinorrhea, headache, or abdominal symptoms
(i.e., diarrhea and vomiting).

Viral RNA was extracted from respiratory samples (Oro-pharyngeal and nasopharyngeal
swabs) using commercially available QIAamp Viral RNA Mini Kit QIAGEN as per manufacturer’s
instructions. For initial detection of Influenza A virus, amplification of matrix
protein (M) gene was carried by real time RT-PCR CDC protocol [13]. For subtyping of Influenza A positive samples, the HA gene (segment 4) of influenza
A(H1N1)pdm09 viruses were analyzed by specific real-time PCR using “Influenza virus
A 1 Real Time RT-PCR Kit” (Shanghai ZJ Bio-Tech Co. Ltd). In order to identify the
Changes in HA amino acid diversity in individual cases, genetic characterisation is
performed by targeted haemagglutinin (HA) sequence analysis and/or partial genome
(931 nucleotide residues) sequencing for a subset of isolates. All viruses analyzed
were amplified and sequenced according to the protocol of National Influenza Centre
Madrid [14]. Primers PHA1+ (5’-GGGGTTAGCAAAAGCAGGRG-3’) and PHA1− (5’-CAWCCRKCIAYCAKICCWKICCAICC-3’)
were used for RT-PCR and H1 + SSEQB (5’-AAYAAYTCIACYGACACTG-3’) and H1-ASEQ (5’-CCCTCAATRAAACCRGCAAT-3’)
for nested PCR.

The sequences were analyzed using the maximum composite likelihood method and the
MEGA version 4.0 software package with 500 bootstrap replicates [15]. Nucleotide sequence accession numbers: The nucleotide sequences of HA determined
in this study can be found in GenBank databases under the indicated accession numbers
JN037697 to JN037779 (http://WWW.ncbi.nih.gov/genomes/FLU/SwineFlu.htmlwebcite).

Ethical approval

The ethical aspects of this study were approved by Charles Nicolle's Hospital ethic
committee.

Results

During the pandemic year, a total of 3836 out of 7350 respiratory specimens with ILI
coming from the sentinel physicians network were positive for influenza A(H1N1)pdm
(95%). In 2010–2011 season, 146 out of 894 of the cases were positive for influenza
A(H1N1)pdm09 (70%). Here we report the occurrence of an amino acid substitution, aspartic
acid to glycine in position 222 (D222G) in the HA subunit of the viral haemagglutinin,
in clinical specimens from 3 out of 42 cases analyzed in Tunisia with severe outcome
(7%). Interestingly, in one fatal case out of four viruses taken from fatal cases
studied (25%). This patient died after 3 days, suffering of severe respiratory symptoms
of flu. Autopsy revealed pulmonary oedema, large mucosis secretions but no cardiac
inflammation (Table 1). Also this mutation was found in one mild case out of 8 mild cases studied (0.1%).
Moreover, D222E was found in one out of 50 viruses studied. This mutation was found
in virus taken from one patient with severe clinical syndrome out of 42 severe cases
studied (2%). E374K substitution was found in two severe cases (4%). This analysis
of HA also showed frequent substitutions in other positions. P83S and S203T were detected
in 94% of Tunisian viruses studied.

Table 1.Characteristics of patients according to the outcome of the infections and molecular
analysis

Discussion

Genetic analysis of HA of influenza A (H1N1)pdm09 virus showed that this virus was
a reassortant containing gene segments from ancestor viruses of human, swine and avian
sources. Hence, Polymorphism at position 222 within the haemagglutinin (HA) molecule
may have remarkable impact on viral host range, replication, and pathogenicity. It
is worth noting that although the Asp222Gly mutation currently has not been associated
with severe pandemic in humans.

An association between D222G and severity was initially proposed by Kilander et al.
(2010) [16] and, since then, different studies in several countries [17] have found the D222G substitution to be more frequently associated with patients
with severe pandemic influenza than in non-severe control cases. A recent study supported
a role for this mutation in allowing the virus access to deeper lung tissue [18]. Hese unusual viral attributes suggested that this new virus may possess some virulence
characteristics similar to the highly pathogenic H5N1 or the 1918 pandemic influenza
viruses.

This study used a conventional sequencing approach to analyze 50 H1N1pdm samples obtained
from 2009 to 2011. Viruses with D222G substitution in HA protein have appeared sporadically
and spontaneously in Tunisia since July 2009 [19]. Although, 7% of them, found in severe cases in the present study, had the D222G
substitution. This percentage is comparable to that found in Italy (4%) [5], in United Kingdom (6%) [17], in France (8%) [20] or in Spain (5%) [14], and lower than that published in Norway (18%) [16], and in Hong Kong (17.4%) [21]. These differences with the Norwegian and Chinese study might be due to the reduced
number of severe cases analyzed in that country. In Tunisia, this mutation was observed
in circulating virus obtained from severe cases [19] and also from mild cases. The frequency of D222G substitution is higher in severe
cases than mild cases. Although most of studies demonstrated the presence of D222G
substitution in severe cases, it was also reported in mild cases [5,22]. Therefore, the number of mild cases would need to be larger to determine whether
mutant viruses are indeed circulating at a very low frequency also in non-severe cases.

The 222 G/E polymorphisms in the haemagglutinin (HA) gene of influenza A(H1N1)pdm09
virus have been associated with cases of mild to severe illness from different countries
or geographical areas [23]. Many retrospective analyses have found that cases bearing the D222G mutation were
more likely to be associated with severe pneumonia, admission to intensive care facilities,
and death [24]. The majority of studies have reported that presence of D222G is sufficient to enhance
virus replication and lethality in mouse models, with this effect ranging from modest
to pronounced [25,26]. Moreover, D222G substitution had been present in the Tunisian virus strain since
pandemic season and throughout 2010–2011 season. Other groups have not observed substantial
differences between wild-type and D222G viruses in mouse or ferret models [6], indicating the need for further investigation into the role of D222G in virulence
of influenza A (H1N1)pdm09. The D222E was detected with less frequency than the D222G
and only found in severe case patient. Despite the available virological, epidemiological
and clinical information, the D222E substitution could confer more severity to the
disease. The clinical significance of this mutation is still unclear [27]. All above studies demonstrated that polymorphism of the HA protein, especially within
the receptor binding domain, play a critical role in the binding preference and pathogenicity
of influenza A(H1N1)pdm09 virus. Further study is warranted to elucidate the intriguing
relationship between D222G substitution and severe disease.

Conclusion

Whether the selection of the D222G mutation is a cause or a consequence of more severe
lower respiratory tract infection is still to be resolved. It is evident, however,
that its emergence is likely to exacerbate the severity of disease. The altered receptor
specificity and distinctive cell tropism of the D222G mutants of influenza A(H1N1)pdm09
are hallmarks of a more dangerous pathogen, emphasizing the importance of close monitoring
of the evolution of these viruses. Influenza A(H1N1)pdm09 variants with 222 G/E polymorphisms
showed increased clinical virulence, and detection of such mutants in the next epidemics
is mandatory for better management of ILI in individual patients as well as for surveillance
purposes especially in African countries.

Consent

For all participants, respiratory samples were collected after informed consent, under
the supervision of local sanitary authorities.

Abbreviations

Competing interests

None of the authors has a financial or personal conflict of interest related to this
study.

Authors’ contributions

AEM: proposed the idea, analyzed and interpreted the data presented in the paper.
Also she wrote the manuscript; FP and JL: participated in the data analysis and interpretation.
Also they revised the manuscript; MTC and IC: revised the manuscript; AS and MHK:
revised the manuscript and save final approval of the version to be published. All
authors read and approved the final manuscript.

Acknowledgements

We are grateful to Ines Laaibi in the National Influenza Centre-Tunis; Mónica Gónzalez-Esguevillas,
Nieves Cruz, Ana Calderón, Noelia Reyes, Manuela Lopez-Valero, Mar Molinero and Silvia
Moreno in the National Influenza Centre Madrid for technical support.